1. Annealing and extension may be accomplished on thermoblocks, or even in metal containers suspended in hot water baths (5), but temperature is critical and must be carefully controlled.

2. In the authors' experience, signal is increased by use of multiple primers for a single locus and by single-step annealing and extension.

3. An unamplified slide without TSAG reagents and an amplified slide without primer should be included as controls for each hybridization.

4. Slides should be kept moist during the PRINS procedure. If a humidified chamber is not available, cover slides with a damp paper towel in a closed box. If a humidifier is available, maintain humidity at 55 to 65% for optimal chromosome spreading (see Subheading 3.5., step 10).

5. Treatment of slides with 0.02 NHCl removes loosely bound protein, thereby rendering DNA more accessible to the primers.

6. For each study, the primer concentration should be optimized. New England Nuclear recommends a 10-fold reduction in "probe" (primer) concentration as optimal. This is a critically important step in PRINS, as improper concentration of probe can obviate development of the hybridization signal.

7. TaqStart monoclonal antibody binds Taq DNA polymerase, thereby minimizing nonspecific amplification and formation of primer dimers.

8. Reagents should completely cover cells or metaphase spreads on slides.

9. After counting the A, C, G, and T nucleotide residues of the primers, annealing temperatures are computed by use of either of the following formulas:

Tm = 69.3 + 0.41 (% G + C) - 650 / L where L = the length of the primer, i.e., the total number of nucleotides in the primer.

When different temperatures are obtained, the results may be averaged. In general, good results are obtained with annealing temperatures between 55°C and 75°C. Higher temperatures increase specificity of annealing.

10. Background staining is minimized by stringent washing of slides in SSC.

11. Innis and Gelfand (6) note that at 20°C, Tris-HCl buffer has a pKa of 8.3, and a A pKa of -0.021/°C. So the actual pH of Tris-HCl may vary during thermal cycling.

12. Low signal may be corrected by titration of HRP conjugate to optimize concentration, by increasing concentration of amplification reagent (or incubation time), or by addition of a step to optimize penetration of reagents (7). Excessive background staining may be reduced by decreasing concentration of HRP conjugate or primers, by increasing endogenous peroxide quenching, by filtration of buffers, or by increasing the number or length of washes (7).


1. Pellestor, F., Girardet, A., Andreo, B., and Charlieu, J.-P. (1994) A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS). Hum. Genet. 94, 346-348.

2. Pellestor, F., Girardet, A., Lefort, G., Andreo, B., and Charlieu, J. P. (1995) Use of the primed in situ labelling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y. Hum. Genet. 95, 12-17.

3. Cinti, C., Santi, S., and Maraldi, N. M. (1993) Localization of single copy gene by PRINS technique. Nucleic Acids Res. 21, 5799-5800.

4. Kadandale, J. S., Tunca, Y., and Tharapel, A. T. (2000) Chromosomal localization of single copy genes SRY and SOX3 by primed in situ labeling (PRINS). Microb. Compar. Genom. 5, 71-74.

5. Koch, J., Hindkjsr, J., K0lvraa, S., and Blund, L. (1995) Construction of a panel of chromosome-specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ. Cytogenet. Cell Genet. 71, 142147.

6. Innis, M. A. and Gelfand, D. H. (1990) Optimization of PCRs, in PCR Protocols, A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic Press, San Diego, CA, pp. 3-12.

7. NEN® Life Science Products, RENAISSANCE ® "TSA™ Biotin System" Laboratory Manual.

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